ANTIBODY COATING APPROACH INVOLVING GAMMA-GLOBULINS FROM NONIMMUNIZEDANIMAL AND 2ND ANTIBODY ANTISERUM

An alternative protocol for immobilization of antibodies onto plastic solid supports is presented. According to the proposed protocol, tubes are first coated with gamma-globulins hom non-immunized animal of the same species as that from which the antigen-specific antibody has bee n developed. Then, an excess of second antibody is added to the tubes and the anti-species specific antibodies present in the antiserum are immunoadsorbed on the immobilized gamma-globulins. Finally, the antige n specific antibody is immunoadsorbed on the immobilized second antibo dy. We found that the coating protocol developed allows the use of ant igen-specific and second antibody antisera dilutions, thus avoiding th e need for affinity purification of antibodies. Additionally, it provi des solid-phase second antibody with increased binding capacity compar ed to the directly adsorbed onto the solid second antibody. The advant ages of the proposed coating protocol were demonstrated through the de velopment of a solid-phase radioimmunoassay for the determination of t otal triiodothyronine in human serum samples.